The agglutination reaction has long been used in qualitative and quantitative assays for a wide variety of bacteria, cell-surface antigens, serum proteins and other analytes of clinical interest. Agglutination usually results from the reaction between antibodies and antigens of interest to produce aggregates which can be detected and/or measured in various ways. In a typical agglutination assay format, particles such as polystyrene microparticles (commonly known as "latex") are coated with a material which binds an antigen of interest, and the coated particles are mixed with a sample, such as blood or other bodily fluid. Depending upon the particular antigen, the interaction may cause the particles to agglutinate with one another, or it may retard agglutination. In either case, the number of agglutinated particles present after the particles are reacted with the bodily fluid will indicate the amount of the antigen present. The same types of reaction have been utilized for the detection of specific antibodies by the agglutination reaction caused by the addition of the corresponding antigen which binds the antibody.
A number of particle-based immunoassays have been developed to exploit the specificity of antigen-antibody reactions, while avoiding the problems associated with conventional diagnostic methodology such as radiochemical labeling. For example, turbidimetric and nephelometric methods monitor light scattered from many particles in bulk solution. The former technique measures light transmission through a suspension of particle aggregates, whereas the latter technique directly measures scattered light in specific directions. These methods offer the advantage of not requiring a separation step; however, they have proven satisfactory only for the analysis of single analytes in a sample.
Several agglutination assays have been developed using optical flow particle analyzers that sense aggregate formation or the degree of non-agglutination, by the measurement of forward scattered light using particles having different sizes. The presence and/or amount of an analyte in a sample has been determined by measuring the extent of agglutination or the number of non-reacted or non-agglutinated particles. The agglutination assay disclosed in U.S. Pat. No. 4,279,617, to Masson et al., involves the steps of mixing a liquid sample with a first particulate reagent which binds the antigen or antibody of interest to form a complex, and then adding to the mixture a second, different particulate reagent which binds to the complex to form an agglutinate, but which does not bind with the free first particulate reagent. The presence or the amount of the antigen or antibody is determined by assaying the non-agglutinated first or second particulate reagent. Thus, to conduct a qualitative assay, the '617 patent teaches that it is merely necessary to count the number of first reagent particles remaining free in the reaction mixture, and then compare the result with the number of such particles added in the first step. In a later published article in Meth. Enzymol. 74:106-141 (1981), Masson et al. coined the acronym PACIA (particle counting immunoassay) to describe agglutination assays that involve counting the number of free or non-reacted particles as an indication of the presence of an analyte in a sample. Masson discloses the use of an AutoCounter to measure forward light scatter from only non-agglutinated particles within a certain size range, to the exclusion of all other particles. The PACIA method, however, has limited use, particularly due to inaccuracies caused by non-specific aggregation reactions and rheumatoid factor interference. See, Newman, et al., Ann. Clin. Biochem. 29:22-42 (1992), and Limet, et al., J. Immunol. Meth. 28:25-32 (1979).
U.S. Pat. No. 4,184,849 to Cambiaso et al. also teaches the use of optical counting system such as an AutoCounter in an agglutination assay, wherein the presence of antibodies or antigens in a liquid is detected by mixing the sample with two different particulate reagents which mutually agglutinate, but which agglutination is inhibited by the particular antibody or antigen being assayed. The amount of antigen or antibody present in the sample may be measured by determining the extent of agglutination or non-agglutination, the latter by simply counting the non-agglutinated particles or by using an identifying label.
U.S. Pat. No. 4,191,739 to Uzgiris, et al., is directed to an agglutination assay wherein the presence of a protein in a sample is determined by detecting multiplets of particles having a size formed by aggregation of only those particles having a first predetermined size with particles having a second predetermined size, using resistive pulse analysis. The method purports to enhance the accuracy of the assay by accounting for the initial distribution of non-specifically formed multiplets.
U.S. Pat. No. 4,851,329 to Cohen, et al., is directed to an agglutination assay wherein the cluster size distribution of the agglutinated particles is determined by optical pulse particle size analysis. A standard quantitative relationship may be established by measuring the ratio of a number of particle dimers to the number of particle monomers, or any other measure of cluster size distribution, in a given serial dilution of known concentration of analyte.
These assay methods are limited to single analyte analysis. Accordingly, there remains a need for an agglutination assay for the simultaneous measurement of a plurality of analytes in a given sample, and which also offers greater precision by reducing the effect of dilution errors, instrumentation flaws, and the like, on the assay results.